FOOD BORNE
BACTERIAL TOXINS
OVERVIEW OF PRESENTATION
 INTRODUCTION
 CHARACTERISTICS OF BACTERIAL ENDOTOXINS AND CLASSIC EXOTOXIN
 PATHOGENESIS BASED EXOTOXINS CLASSIFICATION
 EVENTS OF FOOD BORNE DISEASES
 FOOD BORNE ILLNESS
 ENTEROTOXINS
 PROPERTIES OF HEAT LABILE AND HEAT STABLE ENEROTOXINS
 HEMOLYTIC EXOTOXINS
 NEUROTOXIN
 METHODS FOR DETECTION OF TOXINS.
 SUMMARY
INTRODUCTION
 Toxins are the toxicants or poisonous substance liberated or produced by
living organism and generally not well defined chemically.
Depending on origin
Bacterial toxins
Mycotoxins
Zootoxins
Phytotoxins
Cont…
BACTERIAL TOXIN
ENDOTOXIN
EXOTOXIN
Cont…
ENDOTOXIN
CHARACTERISTICS OF BACTERIAL ENDOTOXINS AND CLASSIC EXOTOXIN
PROPERTY
CHEMICAL NATURE
RELATIONSHIP TO CELL
ENDOTOXIN
LPS (mw = 10kDa)
Part of outer membrane
EXOTOXIN
Protein (mw = 50-1000kDa)
Extracellular, Diffusible
HEAT STABILITY
Yes (600 C)
Relatively No
ANTIGENIC
Yes
Yes
FORM TOXOID
No
Yes
POTENCY
SPECIFICITY
Relatively low (100ug)
Low degree
Relatively high (1 ug)
High degree
ENZYMATIC ACTIVITY
No
Often
PYROGENICITY
Yes
Occasionally
SYNTHESIS CONTROL
Chromosomal gene
Extrachromosomal gene
PATHOGENESIS BASED EXOTOXINS CLASSIFICATION
ENTEROTOXINS
HEMOLYTIC EXOTOXINS
NEUROTOXIN
CYTOTOXIC –CYTOLYTIC
EXOTOXIN
DIRECT MACROMOLECULAR
SYNTHESIS INHIBITOR
V. cholerae, E. coli, B. cereus,
perfringens, Salmonella, Staph. aureus,
Shigella, NAG vibrios, Aeromonas.
Staph. aureus, Streptococcus,
Cl.perfringens, V. parahaemolyticus,
B. cereus, Aeromonas hydrophila.
Cl. botulinum, Cl. tetani,
Shigella dysenteriae.
Strept. Staph. aureus, Shigella ,
Aeromonas, V. parahaemolyticus,
Cl. difficile.
Corynebacterium diphtheriae, E. coli
B.thuringensis, Yersinia pestis,
Pseudomonas, Vibrio cholerae.
Cl.
EVENTS OF FOOD BORNE DISEASES
Reservoir of Pathogen
Viral or Parasitic
infection
Contamination of food
Growth of pathogenic bacteria
Food+ Live cells
Intoxication
Infection
Invasive Infection
Food +Toxin
Toxicoinfection
Mycotoxin
Cont…
Invasive Infection
Toxicoinfection
Intoxication
o Salmonella
o L. monocytogenes
o Bacillus cereus
(Diarrhoel)
o E. coli
( enteric type)
o Shigella
o Campylobacter
o Yersinia
o Vibrio parahaemolyticus
o Aeromonas
o Cl.botulinum
( Infant)
o Staph. Aureus
o B.Cereus
o Cl . perfringens
(Emetic type)
o Vibrio cholarae
o Cl. botulinum
o E. Coli
(Enterotoxigenic)
BACTERIAL
FOOD BORNE
FOOD
BACTERIAL
BORNE INFECTION
ILLNESS
Cont…
FOOD BORNE BACTERIAL ILLNESS
Cont…
FOOD BORNE BACTERIAL ILLNESS
ENTEROTOXINS
 Bacterial toxins that exert some deleterious effect and host response
exclusively in the small or large intestine.
 Alteration in intestinal cell structure or function by o
o
o
A diversity of mode of action,
Target cell types,
Receptors.
Salmonella
 Protein associated with cell wallo Produces fluid loss in infant mice.
 Heat stable and rapid in action.
 Heat labile and delayed response :o Mol. Wt. of 25 kDa,
o Increases in cAMP and PGE2 levels.
o Stimulate secretion in 18-h
Cont…
V. cholerae
 Cholera is caused by V. cholerae of the O1 and O139 serogroups.
CT
Zot
Ace
 CT is the prototypic A-B subunit toxin (A/B ratio, 1:5),
o B is the subunit (11.6 kDa) responsible for binding of the
holotoxin to its GM1 receptor
o A is the subunit responsible for the intracellular
changes in cyclic AMP levels.
Cont…
Mechanism of Action of Cholera Toxin
GM1
Cont…
 If a strain is CT positive, it is almost always zot positive (48 KDa peptide) .
 The onset of action of crude Zot is immediate and reversible.
 Zot may contribute to diarrhea in cholera by altering the permeability of
intestinal tissue.
 Ace 11.3-kDa protein. stimulate electrogenic chloride secretion & contribute,
to the pathogenicity of V. cholerae O1.
NAG Vibrio
 Produce a 17-a.a. NAG-ST that shares 50% sequence homology with the STa
of ETEC .
 This toxin is found only in a minority of non-O1/non-O139 V. cholerae strains.
Cont…
E. coli
 EAECEAST 1
4.1 kDa protein
Heat-labile protein
108 kDa ( Pet)
Chrom.encoded
110 kDa (Pic)
 EIEC –
ShET2 or
EIEC enterotoxin
S. flexneri
 EHEC Shiga toxin
S.dysenteriae
STx, SLT or VT
Stx1, SLT-I or VTI
Stx2, SLT-II or VT2
(Stx2c,2d,2e)
Cont…
ST
LT
 ETEC
STa
o
o
o
o
Cysteine-rich,
18 / 19-a.a. peptide
Mol wt 2 kDa
Bind to G C CIncrease cGMPPKACFTRChloride sec.o Diarrhea
o Children -more
effected
o Human & Animal
STb
o Trypsin-sensitive
protein
o No toxic domain
o Nor Intestinal
receptor
o Sulfatid receptor
o No effect on cAMP
or cGMP.
o Not Stimulate
chloride sec.
o Bicarbonate sec.
o Piglets
LTI
LTII
o Absent of B
subunit
o 2 sub unit
o Mol wt 80,000
LTIIa &LTIIb
o Bind to GM 1
o Bind too Activate
Gangliosid
Adenylate cyclase
GD1b or GD1a
o Increase cAMP
o Increase cAMP
o Increase PG
o Animal disease
o Stimulate GIT
Nervous system
o Human
& Animal
Cont…
Shigella
S. dysenteriae I
Shiga toxin
S. flexneri 2a.
ShET1
o Chomo. Encoded
o Iron dependent
o 55 KDa
ShET2 or
EIEC enterotoxin
o Plasmid encoded
protein
o 63 KDa
Cont…
C. jejuni
o Heat-labile enterotoxin (LT-like)
o 60 to 70 kDa
o Iron regulated.
Y. enterocolitica
o Yst
o Produced at o 30o C
o Increased osmolarity
& pH at 370 C favour Yst.
o Guanylate cyclase activation
o Increase cGMP levels
o Yst-II
o 10- to 30-kDa
o Protein toxin
o Similar to E. coli STa
Cont…
Exotoxins
Staph. aureus
PTS Ags
TSST-1
Hemolysins
SEs
o 18 toxinso Classical enterotoxins SEA to SEE
o Recently discovered –
o True SEs-- SEG, SEH & SEI
o SEls( SElJ to SElP and SElU ).
Cont…
 SEs - thermostable , resistant to GIT enzymes. Heat stability
depend on the environmental factors - aw & pH .
(Balaban et al., 2000)
 SEA and SED, the most frequently involved in food poisoning .
(Balaban et al., 2000 ; Le et al ., 2003 )
 5% cases attributed to new enterotoxins & SEH most commonly
reported .
(Jorgensen et al ., 2005)
 SEs are potent emetic agents whereas the other PTSAgs are not .
 The conc. of SE from 0.5 to 1.0 ng/ml and depends on the
susceptibility of the individual.
Cont…
B. cereus
DIARRHEAGENIC
toxin
o
Thermolabile antigenic
protein
o
Mol wt. 38,000 - 57,000 Da
o
Susceptible to –
o 560 C - 30 min.
o Trypsin & Pronase
digestion
EMETIC
toxin
o Highly stable
o Probably a peptide
o Mol. Wt. < 10,000
o Withstand –
o 1260C - 1.5 hr,
o Extreme pH,
o Typsin & Pepsin
exposure
Cont…
Emetic Form
Diarrheal type
o Wide-range of foods.
o
o
o
o
o Cooked rice & improperly
refrigerated foods .
Large no. of the m.o.
o
Preformed toxin
o
I P 1–5 hours
Diarrhea, abd. cramps
& tenesmus.
o
Nausea & Vomiting
Long-incubation form.
o Recovery within 24 hr
IP 8–16.5 hr.
o Clostridium perfringens
Toxicoinfection
o Staphylococcus aureus
Intoxication
Cont…
Aeromonas
 Several cytotonic enterotoxins
o 15-20 kDa heat
stable protein
.
o
44 kDa heat labile protein
o
CT-like enterotoxin
PROPERTIES OF HEAT LABILE AND HEAT STABLE ENEROTOXINS
Heat Labile
Salmonella, E. coli, Campylobacter ,
Vibrio cholerae, Aeromonas, B.cereus
o
o
o
o
o
PRODUCTION :
o In host
o During vegetative growth
o Secreted in Ileum
o Sub unit 6 subunits (1 A & 5 B )
TRYPSIN
o Proteolysis
Heat stability
o 60 0 C(650 C -30 min.)
ENZYMATIC ACTIVITY
o ADP ribosylate
AFFECT
o Bind to Ganglioside
receptor (epithelialCell)
o Alteration of trans membrane
signal transduction
o Diarrhoea
Heat Stable
E.coli, Y.enterocolitica,
Staph.aureus, B.cereus.
No effect.
1000 C -30 min.
HEMOLYTIC EXOTOXINS
α
β
Streptococcus
SLS
SLO
Cl.perfringens
α
δ
Staph.aureus
V. parahaemolyticus
TDH(1000 C -10 min)
B. cereus
A. hydrophila
Primary hemolysin –
H-1, Cereolysin &
Mouse lethal factor
α hemolysin
2ndry –
H -II
β hemolysin
(Aerolysin)
γ
θ
δ
NEUROTOXIN
:Clostridium
botulinum
 BoNT also known as “Botox”.
 150-kDa zn-binding metallo protease (holotoxin) 100-kDa heavy & 50kDa light chain connected by a reducible disulphide bond .
(Schiavo et al., 2000)
 100,000 times more toxic than sarin .
(Shapiro et al., 1998).
 Estimated human lethal i.v. dose 1-2 ng/kg body weight .
( Arnon et al., 2001)
 Botulin toxin - potential bioweapon, 75 nanograms to kill a person
(LD50- 1ng/kg).
(Fleming., 2000)
 500 grams is enough to kill half of the entire human population
Shigella dysenteriae

Shiga toxin.
METHODS FOR DETECTION OF TOXINS

BIOASSAY METHOD.

IMMUNOLOGICAL ASSAYS.

MOLECULAR TECHNIQUES.

CELL CULTURES.
BIOASSAY METHOD
WHOLE ANIMAL ASSAYS
MOUSE LETHALITY
o Botulinum toxin in foods
o Cl.perfringens enterotoxin
o Listeria virulence assesment
Cont…
FERRETS
o Staphylococcal enterotoxin B
MONKEY FEEDING TEST
o Staph enterotoxins
( Most sensitive )
Cont…
KITTEN / CAT TEST
o Staph enterotoxins
SUNCUS MURINUS EMESIS MODEL
o
Cereulide – B. cereus
Cont…
RABBIT & MOUSE DIARRHOEA
o E.coli
o Y.enterocolitica
o Vibrio cholerae
(enterotoxins)
SUCKLING / INFANT MOUSE
o
E. Coli enterotoxins
( STa, STb )
o
Y. enterocolitica
(Yst)
Cont…
RABBIT SKIN TEST
Vascular permeabilityo
E. Coli
(diarrheagenic enterotoxin)
GUINEA PIG SKIN TEST
Erythemal activityo Cl. perfringens enterotoxin
Cont…
SERENY & ANTON TEST
Guinea pig, Mouse & Rabbit
o
Shigella
o
EIEC
o Listeria
Cont…
ANIMAL MODELS REQUIRE SURGICAL PROCEDURES
LIGATED LOOP TECHNIQUE
o
o
o
o
B.cereus,
Cl perfringens,
E. coli,
V. parahaemolyticus
RITARD MODEL
o
o
o
o
ETEC
V.Cholerae
C. Jejuni
Aeromonas
IMMUNOLOGICAL ASSAYS
ELISA
Double antibody "sandwich" ELISA

Staph enterotoxins A - E
Bennett,.et al; 1994
Cont…
Radioimmunoassay
 Solid phase RIA :
Staph. ET -: A to E
 Sensitivity - 1-5 ng
range
 E.coli STa
Cont…
Reverse passive haemagglutination
 Antibody coupled to sheep RBCs
 Agglutination if toxin present
 Sensitivity for SE–B : 1.5 ng /ml
VIDAS SET Assay
 Staph. enterotoxins A - E
 Sensitivity of at least 1 ng/ml.
Immunoenzymatic test.
 Specific for Staph enterotoxin A - E.
Cont…
Gel diffusion assay
 Microslide gel double diffusion test:

B.cereus diarrhoeal enterotoxins

Cl. perfringens enterotoxins

Staph. B enterotoxin
 Simple gel ID tech. (Biken’s test):
 E. coli
 Sensitivity : 98%
 Time: 3-4 days
Cont…
Electroimmunodiffusion
 SET-A
 B. cereus enterotoxin
 Cl.botulinum enterotoxin A to E.
Cont…
Radial immunodiffusion assay
 Detection of ST- A,B
 Sensitivity –
0.3mcg /ml
Cont…
Reverse passive latex agglutination
 Detection of soluble antigen
 Antibody + latex particle
 Presence of toxin : diffuse layer
at base
 Negative : button formation
 Simple, rapid
 B. cereus emetic toxin.
Cont…
BoNT/A ALISSA
 Assay with a large immuno-sorbent surface area.
 Captures a low number of toxin molecules and measures their intrinsic
metalloprotease activity with a fluorogenic substrate.
 An inexpensive, simple and robust procedure that ensures high analytical
specificity and attomolar sensitivity for the detection of BoNT/A in complex
biological samples
Cont…
ECL ASSAYS
 ELECTRO CHEMI LUMINESCENCE

To detect Clostridium botulinum
neurotoxins serotypes A, B, E, and F

Similar to the gold standard mouse
bioassay.
MOLECULAR TECHNIQUES
PCR
 Duplex SYBR green I real-time
PCR assayfor one-step differentiation
between emetic B. cereus and S.
aureus.
(Mackay., 2004)
 Real-time PCR-based assays BoNT A, B, E and F gene fragments
(Akbulut ., 2005)
Cont…
TaqMan-based, real-time
PCR assay –
 Provide a rapid and
sensitive method for the
specific detection of emetic B.
cereus in food
NUCLEIC ACID PROBES
 Cl.perfringens enterotoxin
 Staphylococcal enterotoxin
CELL CULTURES
Human foetal intestine

V.parahaemolyticus
CHO monolayer
 E.coli LT
 V.cholerae ET
 Salmonella ET
Cont…
Vero cells

E.coli LT

Cl.perfringens ET

Salmonella cytotoxin

A.hydrophilla toxin
Y-1 adrenal cells
 E.coli ET
 V.cholerae toxin
SUMMARY
 Both Gram positive and Gram negative bactera are resposible for
most of the outbreak of the food poisining.
 Bacterial exotoxins have enterotoxic, cytotoxic, hemolytic and
neurotoxic effect .
 It is the bacterial enterotoxins which are responsible for production of
various type of gastrointestinal manifestations like diarrhea and
vomiting during different food borne bacterial illness.
 Some bacterial toxins are very potent and relatively easy to produce
and classified As bio-threat agents. Eg.. Botulinum neurotoxins.
 Bioassay method, Immunological assays, Molecular techniques & Cell
cultures are used to detect the bacterial toxin.
REFERENCES:
www.Slideshare.com